This study was aimed to explore the clinical significance of NK cell activity and serum soluble CD25 (sCD25) level in early diagnosis of the patients with secondary hemophagocytic lymphohistiocytosis (HLH). 38 suspected secondary HLH patients from June 2005 to June 2008 and 25 healthy subjects were enrolled in the study. The NK cell activity in peripheral blood was determined by a released LDH assay, The sCD25 level in serum was detected with ELISA double antibody sandwich assay. The 38 suspected secondary HLH patients were divided into diagnosed and excluded group according to HLH-2004 diagnostic criteria, The NK cell activity and sCD25 level were compared between the two groups. The results showed that 22 out of 38 suspected patients were diagnosed as secondary HLH, the NK cell activity in peripheral blood of these 22 patients was significantly lower than that of healthy control (p < 0.001), the sCD25 level in peripheral blood of these 22 diagnosed patients was higher than that of healthy control (p < 0.05). In conclusion, detection of NK cell activity and sCD25 level may be valuable in the early diagnosis of secondary HLH.